The primary overall objectives are the characterization and identification of passive cotransport pathways for Na, K, and Cl in erythrocytes of humans and sheep. These pathways are widespread, and have prominent functional significance in epithelia and other tissues as well. Human red cells appear to have two separate pathways, one for Na/K/Cl and one for K/Cl, while sheep cells (LK genotype) have only the one for K/Cl. We proposed to study various aspects of the mechanisms of these transport pathways, including their degree of asymmetry, the role of Cl, and their dependence on membrane voltage. We hope to label specifically the K/Cl pathway in sheep cells by taking advantage of an antigen associated with the pathway. Antibodies against this antigen prevent stimulation of transport by N-ethylmaleimide (NEM). This feature should permit us to label the sites with 3H-NEM, identify the protein, and characterize it partially. We plan to study the regulatory mechanisms associated with cotransport by investigating the roles of metabolism, phosphorylation of membrane proteins, interaction of peripheral proteins with the transport pathway, and the relationship of all these and the sensitivity of transport to changes in cell volume. We will also study developmental changes in cotransport, principally in experiments on reticulocytes in culture. We will look at changes in transport and numbers of antigens during maturation. We will determine the role, if any, in maturation of three newly discovered features of sheep reticulocytes: a reticulocyte-specific protein, microtubules, and coated vesicles. We will attempt to modify the process of maturation with various agents, including antibodies and calmodulin inhibitors. In all of the studies in this proposal, we plan to use both alloimmune antibodies and monoclonal antibodies.